The LMNA gene encodes two alternatively spliced products, lamin A and lamin C (Lin and Worman, 1993, J. Biol. Chem. 268:16321-16326). The N-terminal 566 amino acids of lamin A and lamin C are identical with lamin C containing 6 unique amino acids at the C-terminus to yield a protein of 572 amino acids. Lamin A, which is 646 amino acids in length, is generated from a precursor protein, prelamin A, by a series of posttranslational processing steps (Young et al., 2005, J. Lipid Res. Oct 5 electronic publication). The first step in prelamin A processing is farnesylation of a carboxyl-terminal cysteine residue, which is part of a CAAX motif at the terminus of the protein. Next, the terminal three amino acids (AAX) are cleaved from the protein, after which the farnesylcysteine is methylated. Finally, the C-terminal 15 amino acids are enzymatically removed and degraded to form mature lamin A.
Lamin A and lamin C are key structural components of the nuclear lamina, an intermediate filament meshwork underneath the inner nuclear membrane. The lamin proteins comprise N-terminal globular head domains, central helical rod domains and C-terminal globular tail domains. Lamins A and C homodimerize to form parallel coiled-coil dimers, which associate head-to-tail to form strings, and ultimately form the higher-order filamentous meshwork that provides structural support for the nucleus (Muchir and Worman, 2004, Physiology (Bethesda) 19:309-314; Mutchison and Worman, 2004, Nat. Cell Biol. 6:1062-1067; Mounkes et al. 2001, Trends Cardiovasc. Med. 11:280-285).
Hutchinson-Gilford progeria syndrome (HGPS) is a childhood premature aging disease resulting from the production of a mutant form of farnesyl-prelamin A, which cannot be processed to mature lamin A. The accumulation of farnesyl-prelamin A is toxic, inducing misshapen nuclei at the cellular level and a wide range of disease symptoms at the organismal level (e.g., osteoporosis, alopecia, micrognathia and dental abnormalities). HGPS is most commonly caused by a spontaneous mutation in exon 11 of LMNA, which activates a cryptic splice site four nucleotides upstream of the mutation (a cytosine to thymidine substitution at codon 608) (Eriksson et al. 2003, Nature 423:293-298). The pre-mRNA derived from the mutated allele is spliced using the aberrant donor splice site and the correct exon 12 acceptor site, yielding a truncated LMNA mRNA lacking the terminal 150 nucleotides of exon 11. As a result of aberrant splicing, a mutant protein lacking 50 amino acids from the globular tail is produced.
Antisense compounds targeting a selected mRNA or pre-mRNA molecule have proven effective at either reducing total levels of target mRNA through target degradation, or altering the ratio of specific target splice products through occupancy-based mechanisms. Given the role of LMNA in diseases such as HGPS, methods of reducing expression of LMNA mRNA or methods of modulating splicing of LMNA pre-mRNA to eliminate expression of mutant lamin A protein are needed.
A method of controlling the behavior of a cell through modulation of the processing of an mRNA target by contacting the cell with an antisense compound acting via a non-cleavage event is disclosed in U.S. Pat. No. 6,210,892 and U.S. Pre-Grant Publication 2002-0049173.
Kole et al. (WO 94/26887 and U.S. Pat. Nos. 5,627,274; 5,916,808; 5,976,879; and 5,665,593) disclose methods of combating aberrant splicing using modified antisense oligonucleotides which do not activate RNase H.
Scaffidi and Misteli (2005, Nat. Med. 11(4):440-445) disclose a morpholino oligonucleotide used to correct aberrant splicing of LMNA in HGPS fibroblasts.
U.S. Pre-Grant Publication 2005-0059071 discloses mutations in the LMNA gene that cause HGPS and methods of influencing expression of LMNA. Such methods include the use of oligonucleotides and other compounds.
Huang et al. (2005, Human Genet., Oct 6, electronic publication) discuss short hairpin RNA (shRNA) constructs designed to target mutant LMNA pre-mRNA or mature LMNA mRNA to decrease expression of mutant lamin A protein.
Harborth et al. (2003, Antisense Nucleic Acid Drug Dev. 13(2):83-105) discuss LMNA gene silencing using siRNA compounds.
Antisense technology is an effective means for reducing the expression of one or more specific gene products and is uniquely useful in a number of therapeutic, diagnostic, and research applications. Provided herein are antisense compounds for use in modulation of LMNA expression, either through RNase H-dependent cleavage, or by modulation of LMNA splicing. Also provided herein is a method for identifying cis splicing regulatory elements of a selected pre-mRNA, such as LMNA pre-mRNA.